Focus on Ultraviolet-Visible Spectroscopy

Whether its typical samples are liquids, solids, or gases, nearly every laboratory has an ultraviolet-visible spectrometer that serves as a workhorse for dozens of applications. In terms of cost/effort versus capabilities, UV-Vis instruments are arguably the great bargain of lab instrumentation.

Why has a technique more than two centuries old become so popular?

Travis Burt, product manager at Agilent Technologies (Santa Clara, CA), explains that the interaction between light and matter is both fundamental and informative. “Consider how much information the naked eye conveys about a material. Even more so, at an atomic level, photon-induced electronic transitions provide highly specific and highly quantitative information, far greater than is possible with the human eye in terms of sensitivity, wavelength range, and spectral resolution.”

In June 2013, Agilent released the Cary 7000 Universal Measurement Spectrophotometer, which incorporates advances in optics and motion that provide measurement of absolute reflection and transmission at variable angles through unattended operation.

According to Burt, the system is suitable for analyzing optical components, thin films, and other advanced materials.

Sales continue to surge in the chemicals, semiconductors, food, beverage, and pharmaceutical industries, and, of course, at colleges.

What keeps UV-Vis going is its varied application base. “Almost every customer has unique applications,” Talbott observes. “So instead of selling a box, vendors almost have to sell a solution, which becomes the great challenge in this marketplace.”

The story of instrumentation shrinking in size, becoming more portable, is well known to readers of Lab Manager. Fully functional UV-Vis spectrometers have been difficult to shrink to portability for various reasons associated with optical and mechanical limitations.

“The old spectrophotometers resembled coffins,” Talbott reminisces. Shrinking electronics and computers, the move to surface-mount technology, and the resulting diminution of power supplies moved UV-Vis in the right direction. Further improvements and miniaturization came from the emergence of high-quality prisms that accommodate more spectral lines and reduce stray light and dispersion. This advance, in turn, enabled monochromators to shrink as well.

The third significant advance came by way of detector technology. Top-tier UV systems still use photomultiplier tube detectors; but silicone diode arrays—while not as sensitive as PMTs—allow significant size reductions. “Silicon diode arrays eliminate the need for a moving prism, which means motors and other mechanical components associated with rotating the prism are gone,” Talbott says.

While truly handheld, fully functional UV-Vis spectrophotometers remain unavailable, these improvements have brought the size down to “hand portable.”

Such devices always represent a trade-off: spectrophotometers reduced to shoebox size invariably lose resolution and sensitivity, but, according to Talbott, that is not always a serious issue. “The ability to run off a battery, and to bring the unit to the sample, has advantages in many situations over having a supersensitive system with software that does everything and anything.”

That is not to say that quality and portability are totally incompatible. The Shimadzu UV-2600 UV-Vis product fits into a very small footprint but, according to Talbott, has some of the highest absorbance values on the market.

Two possible modes

The availability of UV-Vis detection for microtiter plate formats has been a boon for high-throughput analytical and quality control labs. Readings based on absorbance, fluorescence, luminescence, and transmission, as well as sandwich ELISA-type assays, have greatly expanded analysis capabilities in microliter volumes.

Systems from BioTek Instruments (Winooski, VT) and other vendors have been on the market for some time. UV-Vis-based microplate readers are now routinely paired with robotics and automated liquid handling, to the point of being standard equipment.

By comparison, more standard UV-Vis occurs in cuvettes at volumes as high as 3 milliliters. Reagent and sample savings at typical microplate scale may be greater than 90 percent. The automation component significantly improves throughput and consistency, while freeing up operators to do other things.

“Cuvette-based assays can quantify based on a selected wavelength, but that’s pretty much it,” says Peter Banks, PhD, BioTek’s scientific director. “Microplate readers can do so much more and provide many more options.”

BioTek’s readers also provide a cuvette reading system just in case.

While the 90 percent savings in reagents, buffers, solvents, and samples, which is based on a sample size of 50 to 200 microliters, is significant, we know this is not the limit. Thermo Scientific’s line of NanoDrop products, also based on a UV-Vis reader platform, generates full spectral data on samples in the 0.5-2.0 microliter range.

BioTek’s answer to the demand for ultra-low volumes are the Take3 (16 samples) and Take3 (48 samples) Trio Micro-Volume Plates, which are compatible with the company’s Epoch, Eon, and Synergy Multi-Mode and Hybrid readers, as well as the PowerWaveXS2 reader. That means no dedicated instrument is required. Both plate formats employ 2-microliter “microspots” that reduce sample and reagent usage by as much as 99.9% compared with cuvettes and 99 percent relative to standard microtiter plates.